Protein synthesis represents a major commitment of cellular energy and plays a fundamental role in nearly every aspect of metabolism. It also constitutes a critical step in the control of gene expression. The synthesis of each protein ultimately depends on the relative abundance of its MRNA and its intrinsic translatability, i.e. the capacity of that particular MRNA to interact with components of the translation machinery and be selected for translation. Cellular mRNAs vary over a 100-fold range in their translation efficiency. Additionally, their translation rates depend on the particular growth conditions of the cell. A theoretical treatment by Lodish, subsequently confirmed experimentally, postulates that the spectrum of translated mRNAs varies with the overall rate of protein synthesis. "Weak" mRNAs are outcompeted (i.e. are not translated) by "strong" mRNAs when the rate of translational initiation is reduced. In practice, "weak" mRNAs are not translated when the cell is quiescent; all mRNAs "weak and strong" are translated when the cell is active and capable of proliferation. These correlations should have placed protein synthesis at a pivotal position in pathways of growth control and cell differentiation. Instead, most scientific attention was focused on events occurring at the cell membrane, and then on how signals from the environment are transmitted to the cell nucleus and lead to the expression of previously inactive genes. Yet, it is significant that a surprising number of "weak" mRNAs are those encoding for many protooncogenes, e.g., c-myc, pp60-src, 1ck, mos, c-fos; growth factors, e.g. TGF.beta., FGF, IL-1.beta., Insulin-like GF; growth-related genes, e.g., ornithine decarboxylase, ornithine aminotransferase, and the ribosomal proteins. All of the transcripts mentioned in these groups share the property of being cell-cycle regulated, and their protein products affect cell cycle progression.
Nevertheless, the hypothesis that a link must exist between the rate of protein synthesis and some regulatory function of cell growth has never been formulated. It is only because of very recent, and accidental findings, that these concepts are now emerging.
Because of the central role that protein synthesis has occupied throughout evolution, it is not surprising that translation rates are tightly regulated by some of the most sophisticated mechanisms known. In mammals most of the regulatory mechanisms thus far discovered operate at the step of translation initiation, rather than elongation or termination. The initiation process is envisions as comprising three steps: 1) formation of the 43S complex containing the initiation factors eIF-2, eIF-3, Met-tRNA and GTP, bound to a 40S ribosomal subunit; 2) formation of the 48S complex containing mRNA, which is mediated by the eIF-4 group of factors; 3) formation of the complete 80S complex upon joining of the 60S subunit.
In most circumstances, the regulation that takes place at the second step is rate limiting and specific, because one particular mRNA must be selected and recruited to the ribosomes. As mentioned above, this step is mediated by the eIF-4 group of factors, of which, eIF-4E is by far the least abundant and most likely the rate-limiting. The present inventors have show this experimentally with the application of antisense RNA technology since protein synthesis rates were directly proportional to the level of eIF-4E.
The initiation of translation in eukaryotes can be regulated at the level of 43S complex formation (binding of met-tRNA.sub.i to the 40S ribosomal subunit) and at the level of 48S complex formation (binding of mRNA to the 43S complex). The former occurs during virus infection, following interferon treatment, and in other severe and stressful circumstances. Under more normal cellular conditions, the formation of the 48S complex is rate limiting, and regulation by mitogens, growth factors, serum or during mitosis appears to occur at this step. mRNA binding to 43S complexes is catalyzed by the eIF-4 group factors, which collectively recognize the 7-methylguanosine-containing cap, melt mRNA secondary structure beginning from the 5' end, and facilitate the scanning of the mRNA sequence for the initiation codon by the 40S subunit.
Prior to the present invention, it was not completely understood how mRNA recruitment into 48S initiation complexes is regulated. A factor which is likely to be involved is eIF-4E, a 25-kDa polypeptide which binds to the cap (presumably the first step in mRNA recruitment) and accompanies mRNA transfer to the 48S complex. Whether eIF-4E acts as a free polypeptide, in a complex with other polypeptides, or both, has not been established. eIF-4E is the least abundant of the initiation factors and is present at approximately one-tenth the molar concentration of mRNA and ribosomes.
eIF-4E is a phosphoprotein, the major site of in vivo phosphorylation being Ser-53. Phosphorylation of eIF-4E is correlated with elevated protein synthesis in reticulocytes treated with phorbol esters, fibroblasts treated with serum, B lymphocytes activated with phorbol esters and ionomycin or lipopolysaccharide and 3T3-L1 fibroblasts treated with insulin. Conversely, dephosphorylation of eIF-4E is correlated with the inhibition of protein synthesis in HeLa cells after heat shock or during mitosis. Furthermore, a variant of eIF-4E in which Ser-53 is replaced with Ala-53 (eIF-4E.sup.Ala) cannot be phosphorylated at the major in vivo site and is not found on the 48S initiation complex, suggesting that eIF-4E cannot participate in the transfer of mRNA to the 48S complex unless it is phosphorylated.
It was shown that when eukaryotic cells were transformed with a vector expressing the eIF-4E polypeptide factor (wild type), deleterious effects take place in the cell. In some cases, the expression of the eIF-4E factor was shown to be lethal to the cells whereas analogous cells containing the eIF-4E.sup.Ala variant were not (De Benedetti, A, et al, "Mammalian Expression Vectors for the in vivo Study of eIF-4E", Abstr., p.218, Translational Control, Cold Spring Harbor Laboratory, New York (1989). In another study, De Benedetti et al. (DeBenetti, A. and Rhoads, R. E., "Over-expression of eIF-4E from an Episomal Vector in HeLa Cell Results in Abnormal Growth and Ultimately Cell Death", Abstract 2314, FASEB Journal 4, A2093 (1990)), showed that when the eIF-4E gene is expressed from an episomal system, the wild type overexpresses the factor and accelerates cell growth and division as well as the formation of multi-nucleated cells. This was not the case when cells were transformed with a vector carrying the eIF-4E variant lacking the major phosphorylation site (wild type: SER-53, variant: Ala-53).
Fagan et al., Journ. of Biol. Chem., Vol 266, No. 25, Sep. 5, 1991, p. 16518-16523 disclose an analysis of eIF4E mRNA in each of two different strains of retinoblastomas and its influence on the amount of ornithine aminotransferase in each of the strains of retinoblastomas. This publication does not disclose a hybrid vector according to the present invention.
Koromilas et al., EMBO Journal, Vol 11, No. 11, pp. 4153-4158 (1992) disclose that eIF4E overexpression facilitates the translation of mRNAs with 5' untranslated region (UTR) extensive secondary structures. This publication does not disclose a hybrid vector according to the present invention.
Shatzman and Rosenberg in Methods of Enzymology, Vol. 152 (1987) disclose the Shine-Delgarno sequence of prokaryotic messenger RNA. The Shine-Delgarno sequence provides an alignment between the mRNA and the 18S rRNA. This is important in the context of the correct positioning for translation initiation, but does not increase the rate of ribosome-binding of a particular mRNA. In eukaryotes, "stored" untranslated mRNAs can, at anytime, be recruited for translation, a reaction mediated by eIF4E. Thus, the knowledge that the binding of eIF4E to the 40S ribosomal subunit (mediated by eIF4E) is the rate limiting step for translation initiation, under normal conditions which was discovered by the present inventors and is distinct from prokaryotic translation initiation functions.